An increase in the extracelluar concentration of the neurotransmitter glutamate, as seen in hypoxia-ischemia, results in neuronal damage. Glutamate is most likely released from the presynaptic neuron and, possibly surrounding glial cells, during a hypoxic-ischemic event. The exact mechanism by which glutamate is released into the neurosynaptic junction remains to be elucidated, although there is published evidence to implicate reverse function of an amino acid transporter. Our global hypothesis is that an ASCT amino acid transporter contributes to glutamate-mediated excitotoxicity observed in hypoxic-ischemic states by catalyzing glutamate efflux into the neurosynaptic junction only at the lower pH values associated with pathological conditions, such as hypoxic ischemic encephalopathy (HIE). ASCT1 is a neutral amino acid transporter at physiologic pH, but at lower pH shifts its substrate specificity to accept anionic amino acids such as glutamate. These lower pH values would exist during HIE. The immediate goal of the experiments outlined is to test this hypothesis by determining if ASCT1, by itself or with other glutamate transporters, is responsible for the efflux of glutamate under hypoxic- ischemic conditions. In vitro model systems will include investigation of pH-dependent D-aspartate (a glutamate analog with regard to transport) efflux from primary cultures of neonatal neurons. In addition, overexpression of both anionic amino acid transporters and ASCT transporters in HeLa cells to document which of these transporters is capable of mediating pH-dependent D-aspartate efflux. Secondly, we will use both in situ hybridization and immunohistochemistry to document the expression of the ASCT1 transporter protein after hypoxic-ischemic events, both in neuronal cultures in vitro and in intact brain tissue in vivo, to gain a understanding of its possible modulation by biosynthesis or degradation following neuronal injury.